Disproportional Distribution of Isotype and Non-lsotype-Specific IgG Subclass Anti-lgE Autoantibodies in Human Cord Serum

Abstract

The levels of naturally occurring IgG and IgG subclass anti-IgE autoantibodies (a-E Ab) were studied in 71 randomly collected cord sera with ELISA using solid-phase IgE-DES myeloma protein. IgG a-E Ab were present in all cord sera, and the range was 300-fold (1.8–540 arbitrary units/ml; median =11.8). However, this acticity is the sum of two major types of a-E Ab that we refer to as isotype-specific (IS) and non-isotype-specific (NIS) because they react with E-chain-specific and myeloma-restricted epitopes, respectively. These two types of a-E Ab were distinguished in IgG subclass a-E Ab inhibition ELISA for all samples using unheated IgE-PS and heated IgE-DES as inhibitors. IS and NIS a-E Ab were found among all four IgG subclasses though with different prevalences. No significant influence of gender was observed. Comparisons within each subclass indicate that NIS a-E Ab were more common than IS a-E Ab for the IgG1 (p < 0.00005), IgG2 (p = 0.07) and IgG3 (p < 0.005) subclasses while the reverse was true for IgG4 (p < 0.00005). In fact, only a minor part of the IgG1 (7%), IgG2 (13%) and IgG3 (9%) a-E Ab activity towards IgE-DES was IS while for IgG4 it was a major part (82%). Attempts to quantify IS and NIS IgG subclass a-E Ab using chimeric IgG subclass antihapten Ab suggested that the pool of IgG a-E Ab against IgE-DES was dominated by NIS a-E Ab particularly of the IgG1 subclass. The 75 percentiles for NIS IgG1, IgG2, IgG3 and IgG4 a-E Ab were 24, < 2, 2.1 and 0.27 ng/ml, respectively, whereas the corresponding figures for IS a-E Ab were 7, < 2, < 2 and 0.90 ng/ml. The findings raise questions on the definition of a-E Ab and suggest that caution should be exercised in the interpretation of any a-E Ab results that are detected with IgE myeloma proteins. The potentially more interesting IS a-E Ah may be overshadowed by the bulk of ubiquitous NIS a-E Ab and background. Consequently, discriminatory assays are necessary if the physiological implication of naturally occurring IS IgG subclass a-E Ab is to be elucidated and these considerations are not limited to studies of cord serum.

Vaccination with Ep-CAM Protein or Anti-Idiotypic Antibody Induces Th1-Biased Response against MHC Class I- and II-Restricted Ep-CAM Epitopes in Colorectal Carcinoma Patients

“…Ep-CAM-specific IgG1, IgG2, IgG3, and IgG4 Abs were determined as described previously (28). The coating concentration for Ep-CAM and gp160 was 2 g/ml.…”

“…High-titered sera were further assayed at higher dilutions. The Ab concentrations were calculated from standard curves established from chimeric IgG1, IgG2, IgG3, and IgG4 anti-5-iodo-4-hydroxy-3-nitrophenacetyl acid hapten Abs using bovine serum albumin (BSA)-5-iodo-4-hydroxy-3-nitro-phenacetyl acid conjugate (10 g/ml) as coating Ag (28). Taking serum dilution into account and correcting for background, the sensitivity of the assay was 8 (IgG1), 4 (IgG2 and IgG3), and 0.4 EU/ml (IgG4).…”

“…After washing, antihuman IgG1 mAb (1:5000 dilution; Ref. 28) was added for 6 h at room temperature, followed by alkaline phosphatase-conjugated rabbit F(abЈ) 2 antimouse IgG Ab overnight at 4°C (28). After washing in 0.9% NaCl, the membranes were developed with alkaline phosphatase color buffer (Bio-Rad Lab, Stockholm, Sweden).…”

IgG1, IgG4 and IgE Antibody Reactivity to Mutant Forms of the Major Dust Mite Allergen Lep d 2 among Atopic and Nonatopic Subjects Naturally Exposed to <i>Lepidoglyphus destructor</i>

“…Allergen-specific IgG1, IgG4 and IgE Ab were determined by sandwich ELISA using monoclonal Ab essentially as previously described [20, 21]. Briefly, microtiter plate wells were coated with purified allergens (5 µg/ml).…”

“…All serum samples were assayed at a final 50-fold (IgG1), 10-fold (IgG4) or 5-fold (IgE) dilution following preincubation with the corresponding allergen (10 µg/ml) or diluent alone. The net results in optical density at 405 nm were used to interpolate the concentrations of Ab from standard curves of chimeric IgG1, IgG4 and IgE anti-NIP hapten Ab run in parallel using BSA-NIP conjugate (10 µg/ml) as coating antigen [20]. Taking the serum dilution into account and considering a net optical density of 0.020 as cutoff, the serum sensitivity in these assays were 4.0 (IgG1), 0.30 (IgG4) and 0.20 (IgE) arbitrary units (AU)/ml.…”